Professor Hubert Hondermarck

Purpose: Given that nerve growth factor has previously been shown to be involved in breast cancer progression, we have tested here the hypothesis that the other neurotrophins (NT) are expressed and have an influence in breast tumor growth. Experimental Design: The expression of brain-derived neurotrophic factor (BDNF), NT-3 and NT-4/5, as well as the neurotrophin receptor p75 NTR, TrkB, and TrkC, was studied by RT-PCR, Western blotting, and immunohistochemistry in cell lines and tumor biopsies. The biological impacts of neurotrophins, and associated mechanisms, were analyzed in cell cultures and xenografted mice. Results: BDNF and NT-4/5 were expressed and secreted by breast cancer cells, and the use of blocking antibodies suggested an autocrine loop mediating cell resistance to apoptosis. The corresponding tyrosine kinase receptor TrkB was only rarely observed at full length, whereas the expression of TrkB-T1, lacking the kinase domain, as well as p75NTR, were detected in all tested breast cancer cell lines and tumor biopsies. In contrast, NT-3 and TrkC were not detected. SiRNA against p75NTR and TrkB-T1 abolished the antiapoptotic effect of BDNF and NT-4/5, whereas the pharmacological inhibitors K252a and PD98059 had no effect, suggesting the involvement of p75NTR and TrkB-T1, but not kinase activities from Trks and MAPK. In xenografted mice, anti-BDNF, anti-NT-4/5, anti-p75 NTR, or anti-TrkB-T1 treatments resulted in tumor growth inhibition, characterized by an increase in cell apoptosis, but with no change in proliferation. Conclusion: BDNF and NT-4/5 contribute to breast cancer cell survival and can serve as prospective targets in attempts to inhibit tumor growth. ©2011 AACR.

Purpose: We have recently shown that breast tumors express high levels of TrkA compared with normal breast tissues, with TrkA overexpression enhancing breast cancer cell invasion in vitro and metastasis in animal models. In this study, we tried to identify molecules involved in TrkA overexpression-mediated biological effects in breast cancer cells. Experimental design: We used a proteomic-based approach to identify proteins involved in TrkA overexpression-stimulated invasion of MDA-MB-231 breast cancer cells. Proteins from control and TrkA overexpressing cells were separated using a cup-loading two-dimensional electrophoresis system before MALDI and LC-MS/MS mass spectrometry analysis. Results: Among several putative regulated proteins, Ku86 was found increased in TrkA overexpressing cells. Moreover, Ku86 was co-immunoprecipitated with TrkA, suggesting the interaction of these two proteins in TrkA overexpressing cells. Interestingly, inhibition with small-interfering RNA and neutralizing antibodies showed that Ku86 was required for TrkAstimulated cell invasion. Conclusions and clinical relevance: These data allowed the identification of Ku86 as a new player involved in metastasis in breast cancer cells. Our findings suggest that TrkA and its down stream signaling pathways should be regarded as potential new targets for the development of future breast cancer therapy. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Blood vessel formation (neovascularization) in tumors can occur through two mechanisms: angiogenesis and vasculogenesis. Angiogenesis results from proliferation and sprouting of existing blood vessels close to the tumor, while vasculogenesis is believed to arise from recruitment of circulating cells, largely derived from the bone marrow, and de novo clonal formation of blood vessels from these cells. Increasing evidence in animal models indicate that bone marrow-derived endothelial precursor cells (EPC) can contribute to tumor angiogenesis. This review aims to collate existing literature and provide an overview on the current knowledge of EPC involvement in breast cancer angiogenesis. We also discuss recent attempts to use EPC as biomarker and therapeutic target in clinical trials. © 2009 Springer Science+Business Media, LLC.

The p75 neurotrophin receptor (p75NTR) plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation and proliferation. To evaluate the influence of p75NTR in breast cancer development, we have established and characterized breast cancer cells which stably overexpress p75NTR. We showed that p75NTR overexpression per se promoted cell survival to apoptogens with a concomitant slowdown of cell growth. The pro-survival effect is associated with an increased expression of the inhibitor of apoptosis protein-1 (c-IAP1), a decrease of TRAIL-induced cleavage of PARP, procaspase 9 and procaspase 3, and a decrease of cytochrome C release from the mitochondria. The anti-proliferative effect is due to a cell accumulation in G0/G1, associated with a decrease of Rb phosphorylation and an increase of p21waf1. Interestingly, inhibition of p21waf1 with siRNA not only restores proliferation but also abolishes the pro-survival effect of p75NTR, indicating the key role of p21waf1 in the biological functions of p75NTR. Finally, using a SCID mice xenograft model, we showed that p75NTR overexpression favors tumor growth and strongly increases tumor resistance to anti-tumoral treatment.Together, our findings suggest that p75NTR overexpression in breast tumor cells could favor tumor survival and contribute to tumor resistance to drugs. This provides a rationale to consider p75NTR as a potential target for the future design of innovative therapeutic strategies. © 2010 Elsevier Inc.

The p53 tumor suppressor protein is a key regulator of cell cycle and death that is involved in many cell signaling pathways and is tightly regulated in mammalian cells. Post-translational modifications of p53 have been investigated previously mainly using antibodies. In this study, utilizing LC-MS/MS analysis, we have characterized p53 protein from COS-1 cells. Several already known posttranslational modifications were observed, such as phosphorylation on serines 15, 33, 315, and 392 as well as acetylation on lysines 305, 370, 372, 373, 381, 382, and 386. Interestingly novel acetylation sites were identified at lysines 319 and 357. This study confirmed that p53 is a highly acetylated protein and revealed new acetylation sites that might aid the further understanding of p53 regulation. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

We have used a combination of SDS-PAGE and LTQ-Orbitrap MS to explore the proteome of the highly invasive MDA-MB-231 breast cancer cell line. Based on about 520 000 MS/MS spectra, a total of 3481 proteins were identified and subsequently classified according to their cellular distribution and molecular function. Interestingly, a large proportion of proteins (38%) were from cellular membranes and we were able to characterize numerous proteins involved in cancer initiation and progression such as the tumor suppressor p53 and the epidermal growth factor receptor. Together, this study represents the largest proteome database of breast cancer cells realized to date and demonstrates the value of using Orbitrap MS for deeper proteome analysis. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Trk family of neurotrophin tyrosine kinase receptors is emerging as an important player in carcinogenic progression in non-neuronal tissues. Here, we show that breast tumors present high levels of TrkA and phospho-TrkA compared to normal breast tissues. To further evaluate the precise functions of TrkA overexpression in breast cancer development, we have performed a series of biological tests using breast cancer cells that stably overexpress TrkA. We show that (1) TrkA overexpression promoted cell growth, migration and invasion in vitro; (2) overexpression of TrkA per se conferred constitutive activation of its tyrosine kinase activity; (3) signal pathways including PI3K-Akt and ERK/p38 MAP kinases were activated by TrkA overexpression and were required for the maintenance of a more aggressive cellular phenotype; and (4) TrkA overexpression enhanced tumor growth, angiogenesis and metastasis of xenografted breast cancer cells in immunodeficient mice. Moreover, recovered metastatic cells from the lungs exhibited enhanced anoikis resistance that was abolished by the pharmacological inhibitor K252a, suggesting that TrkA-promoted breast tumor metastasis could be mediated at least in part by enhancing anoikis resistance. Together, these results provide the first direct evidence that TrkA overexpression enhances the tumorigenic properties of breast cancer cells and point to TrkA as a potential target in breast cancer therapy. © 2009 Macmillan Publishers Limited All rights reserved.

Proteomics of breast cancer has already delivered significant data in terms of proteome profiling in addition to the identification of a few proteins of potential interest for diagnosis and treatment. With more pathological and experimental situations being studied, it now enters into a new phase dominated by the concepts of deep proteome analysis and the definition of protein-protein interaction networks leading to mammary cell deregulation and cancer progression. Together, what could be called "Systems Proteomics", integrating with information from the genomics and the physiopathology, is clearly emerging to become the frame for future investigations. However, difficulties ahead should not be underestimated. First, the proteome is complex, and current tools are still far from providing a definitive solution for its exploration. Second, breast cancer is a multifactorial disease which is so diverse that a great deal of time and efforts will be necessary to define its associated proteome modifications and translate it into practical applications for the clinic. © 2008 American Chemical Society.

We show here that nerve growth factor (NGF), the prototypic neurotrophin, can be targeted in breast cancer to inhibit tumor cell proliferation, survival, and metastasis. Analysis of a series of biopsies revealed widespread expression of NGF in the majority of human breast tumors, with anti-NGF immunoreactivity concentrated in the epithelial cancer cells. Moreover, immunodeficient mice xenografted with human breast cancer cells and treated with either anti-NGF antibodies or small interfering RNA against NGF displayed inhibited tumor growth and metastasis. Such treatments directed against NGF induced a decrease in cell proliferation with a concomitant increase in apoptosis of breast cancer cells and an inhibition of tumor angiogenesis. Together, these data indicate that targeting NGF in breast cancer may have therapeutic ramifications. ©2008 American Association for Cancer Research.

The H19/IGFf2 locus belongs to a large imprinted domain located on human chromosome 11p15.5 (homologue to mouse distal chromosome 7). The H19 gene is expressed from the maternal allele, while IGF2 is paternally expressed. Natural antisense transcripts and intergenic transcription have been involved in many aspects of eukaryotic gene expression, including genomic imprinting and RNA interference. However, apart from the identification of some IGF2 antisense transcripts, few data are available on that topic at the H19/IGF2 locus. We identify here a novel transcriptional activity at both the human and the mouse H19/IGF2 imprinted loci. This activity occurs antisense to the H19 gene and has the potential to produce a single 120-kb transcript that we called the 91H RNA. This nuclear and short-lived RNA is not imprinted in mouse but is expressed predominantly from the maternal allele in both mice and humans within the H19 gene region. Moreover, the transcript is stabilized in breast cancer cells and overexpressed in human breast tumors. Finally, knockdown experiments showed that, in humans, 91H, rather than affecting H19 expression, regulates IGF2 expression in trans. Copyright © 2008, American Society for Microbiology. All Rights Reserved.